Bioorthogonal reactions for coupling materials in the presence of complex biological milieu are of great interest in biology and medicine. The term refers to reactions between two molecules which, although they react with each other, do not react with the molecules present in living organisms and the functional groups present in biomolecules, and thus can be carried out without interfering with biological processes. Such reactions have become key components in a variety of applications including protein engineering, immunoassay development, and cell surface modification. See, e.g., Baskin et al., Proc. Natl. Acad. Sci. USA, 2007, 104, 16793-97; Best, Biochemistry, 2009, 48(28). pp. 6571-84; Chen et al., Acc. Chem. Res., 2011, 44(9), 762-73; Dimandis et al., Clin. Chem., 1991, 37, 625-36; Kolb et al., Angew. Chem. Int. Ed., 2001, 40, 2004-21; Link et al., Curr. Opin. Biotechnol., 2003, 14, 603-09; Link et al., J Am. Chem. Soc., 2003, 125, 11164-65; Prescher et al., Nature, 2004, 430(7002), 873-77; Prescher et al., Nat. Chem. Biol., 2005, 1(1), 13-21; Lim et al., Chem. Commun. (Camb.), 2010, 46(10), 1589-600; Sletten et al., Angew. Chem. Int. Ed., 2009, 48(38); 6974-98; Wang et al., J. Am. Chem. Soc., 2003, 12, 3192-3193. Presently, a few types of bioorthogonal reactions have been reported.
One type of reaction that has been used is the Staudinger ligation between phosphines and azides. Prescher et al., Nature, 2004, 430(7002), 873-77; Saxon et al., Science, 2000, 287(5460), 2007-10.
Another useful reaction is the [3+2] cycloaddition “click” reaction between azides and alkynes. Rostovtsev et al., Angew. Chem. Int. Ed., 2002, 41(14), 2596-2599. While this reaction proceeds in the presence of copper, a copper-free variant has been developed that does not require the use of copper, involving cycloaddition of azides to a strained alkyne such as a cyclooctyne ring, a dibenzocyclooctyne ring, an azadibenzocyclooctyne ring, or a bicyclononyne (e.g., bicyclo[6.1.0]nonyne) ring. Agard et al., J. Am. Chem. Soc., 2004, 126 (46), 15046-47; Baskin et al., Aldrichimica Acta, 2010, 43(1), 15-23; Cenoweth et al., Org. Biomol. Chem., 2009, 7, 5255-58; Dommerholt et al., Angew. Chem. Int. Ed., 2010, 49, 9422-25; Jewett et al., J. Am. Chem. Soc., 2010, 132 (11), 3688-90; Marks et al., Bioconjugate Chem., 2011, 22(7), 1259-63; Sletten et al., Acc. Chem. Res., 2011, 44(9), 666-76.
Bioorthogonal “click” chemistries are widely used in chemical biology for a myriad of applications such as activity based protein profiling, crosslinking of proteins, monitoring cell proliferation, generation of novel enzyme inhibitors, monitoring the synthesis of newly formed proteins, protein target identification, and studying glycan processing. Bioorthogonal chemistry has been used, e.g., to assemble molecules in the presence of living systems such as live cells or even whole organisms. Baskin et al., Proc. Natl. Acad. Sci. USA, 2007, 104, 16793-97; Laughlin et al., Science, 2008, 320, 664-67; Prescher et al., Nat. Chem. Biol., 2005, 1, 13-21; Neef et al. Angew. Chem. Int. Ed. 2009, 48, 1498-500; Ning et al., Angew. Chem. Int. Ed, 2008, 47, 2253-55. However, to date, the application of “click” chemistry in living systems, has been largely limited to extracellular targets and no technique has shown reliable ability to specifically label and image intracellular targets. Baskin et al., QSAR Comb. Sci., 2007, 26, 1211-19. There are likely several reasons for this limitation. In addition to fulfilling the stability, toxicity, and chemoselectivity requirements of “click” chemistry, intracellular live cell labeling requires reagents that can easily pass through biological membranes and kinetics that enable rapid labeling even with the low concentrations of agent that make it across the cell membrane. Additionally, a practical intracellular bioorthogonal coupling scheme would need to incorporate a mechanism by which the fluorescent tag increases in fluorescence upon covalent reaction to avoid visualizing accumulated but unreacted imaging probes (i.e., background). This “turn-on” would significantly increase the signal-to-background ratio, which is particularly relevant to imaging targets inside living cells since a stringent washout of unreacted probe is not possible.
In previous years a number of elegant probes have been introduced whose fluorescence increases after azide-alkyne cycloaddition or Staudinger ligation coupling reactions. Hangauer et al., Angew. Chem. Int. Ed. Engl., 2008, 47, 2394-97; Lemieux et al., J Am. Chem. Soc., 2003, 125, 4708-09; Sivakumar et al., Org. Lett., 2004, 6, 4603-06; Zhou et al., J. Am. Chem. Soc., 2004, 126, 8862-63. Most of these strategies either require a reactive group intimately attached to the fluorophore thus requiring synthesis of new fluorophore scaffolds or take advantage of a FRET based activation requiring appendage of an additional molecule that can act as an energy transfer agent. Furthermore, most probes utilizing these popular coupling schemes have to date been unable to label intracellular targets in live cells.
The bioorthogonal Diels-Alder reaction is compatible with aqueous environments and has second order rate constants that are known to be enhanced up to several hundred-fold in aqueous media in comparison to organic solvents. Graziano, J. Phys. Org. Chem., 2004, 17, 100-01; Rideout et al., J. Am. Chem. Soc., 1980, 102, 7816-17; Seelig et al., Tetrahedron Lett., 1997, 38, 7729-32; Yousaf et al., J. Am. Chem. Soc., 1999, 121, 4286-87. Many Diels-Alder reactions are reversible and therefore may not be suitable for biological labeling. Kwart et al., Chem. Rev. 1968, 68, 415-47.
A particularly useful variant of the Diels-Alder reaction employs the inverse electron demand Diels-Alder cycloaddition of olefins with 1,2,4,5-tetrazines results in irreversible coupling giving dihydropyridazine products as shown in Scheme 1.

During this reaction, nitrogen is released in a retro Diels-Alder step resulting in an irreversible reaction. Sauer et al., Chem. Ber., 1965, 998, 1435-45. A variety of 1,2,4,5-tetrazines and dienophiles including cyclic and linear alkenes or alkynes have been studied in this reaction. Selection of the appropriate reaction partners, allows for tuning of the coupling rate by several orders of magnitude. The reaction can occur rapidly and at ambient temperatures with a strained alkene or alkynes such as a trans-cyclooctene group or cyclooctyne as a dienophile. Balcar et al., Tetrahedron Lett., 1983, 24, 1481-84; Blackman et al., J. Am. Chem. Soc. 2008, 130, 13518-19; Thalhammer et al., Tetrahedron Lett., 1990, 47, 6851-54). See also US 2006/0269942, WO 2007/144200, and US 2008/0181847, US2009/0023916; US2011/0268654 and US2012/0034161.
Application of bioorthogonal coupling technology is described in WO2010/051530. The publication describes materials and methods for delivering a substance specifically to a biological target by applying the inverse electron demand Diels Alder reaction. In general, the method described involves use of a ligand that is specific for a biological target and a substance which is to be brought into proximity with the biological target. The substance can be, e.g., a detectable substance so that the method can be used for diagnostic applications or a therapeutic substance. The ligand and the substance that is to be delivered are brought into proximity by means of a complementary diene (e.g., a 1,2,4,5-tetrazine) attached to one of the components and a dienophile (e.g., a trans-cyclooctene) attached to the second component. The inverse-electron-demand Diels-Alder reaction between the Diels-Alder components, e.g., the 1,2,4,5-tetrazine and a suitable dienophile, e.g., a trans-cyclooctene, serves to bring the components into proximity.
One of the limitations on the broad applicability of the inverse-electron-demand Diels Alder reaction is that relatively few functionalized 1,2,4,5-tetrazines that are suitable for attachment or incorporation into biological molecules, their ligands, or suitable therapeutic or diagnostic molecules are known in the art.